多個序列突變的篩查HRMA（四）

替代凝膠電泳

    標準是使用瓊脂糖凝膠電泳和EB著色。通過片段的長度、純度和條帶的熒光量確定片段。HRMA是一種很有優勢的技術；通過熔解圖、存在其它熔解圖的純度（沒有額外的熔解峰）和產生的大量熒光信號（圖6）。使用HRMA的優勢很明顯；不需要灌膠、使用危險化學藥品（EB），熔解比電泳速度快，數據分析可以自動完成。此外，HRMA是一種非破壞性的方法，當HRMA沒能得到明確的結果時，片段依然可以在凝膠上進行分析。

圖6.用HRMA替代凝膠電泳。A：5個不同PCR片段的分析；因為片段間有清晰的熔解圖差異，所有五個片段可以在一個分析完成。當熔解圖部分重疊，可以做到每個片段分析。B:PCR上可以進行96個不同樣品的分析。一個PCR失敗的熒光，其它的可以通過熒光水平估計。純化，包括引物二聚體的存在，可以被HRMA差異圖的分析檢測出（未顯示）。C：HRMA后插入384板PCR后，進行第二輪選擇。幾個清晰的熔解組確定，表示包含克隆的組有相同的插入。確認所有組的存在，第一次分析后確認分析需要分離和軟件必須重新分組。重復這一過程直到所有組得到確定。

    在我們實驗室，HRMA迅速取代了凝膠電泳用于分析PCR產物。加入顏料后進行PCR（每10 μL體系加1 μL 10ⅹLC-Green染料），樣品在管內95℃ 5min，降至室溫，然后熔解。圖5顯示了分析5個不同片段，都得到了清晰的熔解圖。在熔解峰低Tm值處可以辨認處引物二聚體。OUT等（提交手稿）使用HRMA代替凝膠電泳檢測擴增，同時在MUTYH基因測序前確定長期的PCR指導。作者成功地檢測了幾乎所有的期望突變達0.5%（1/200同源染色體）

克隆鑒定

    一些實驗產生一系列克隆需要測序確定。這些包括體外突變實驗、甲基化研究、CDNA克隆確定不同剪接的水平或等位基因表達和噬菌體篩選。HRMA提供了一種有吸引力的工具篩選克隆，檢測那些具有相同插入和變異。減少了大量的測序工作。圖6C顯示了第二次噬菌體篩選的結果。幾個克隆組的實驗結果具有相同的熔解圖，顯示實驗成功地選擇了幾個不同的噬菌體克隆。隨后每組的克隆代表測序驗證了HRMA的結果；組內序列差異和測序確定[Pepers等2009]。以前，克隆插入確定是使用酶切和凝膠電泳，靈敏度低且實驗繁瑣[Verheesen 等, 2006]

結論

    HRMA的優勢使它迅速吸引了一批新用戶。HRMA操作簡單、簡單、靈活、成本低，非破壞性、靈敏度和特異性高的特點是它成為了篩查患者致病突變的一種新方法。綜上所述，HRMA有多個吸引力的其他應用，使它具有多種分析核酸的功能。由于HRMA依然是一項新技術，期待它有更好的發展。Fluidigm公司推出了一個納升qPCR系統[Spurgeon等, 2008],目前推出了一個96個樣品分析的PCR實驗(例如，同時進行9261個實驗)；設想這樣一個系統時，將有助于HRMA。

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